1. Using molecular modeling, CD and NMR spectroscopy.[unreadable] [unreadable] The three-dimensional structures of NK-2 class homeodomain proteins, the human NKX 2-1, NKX 2-5, and NKX 3-1 proteins, have been investigated by Dr. Gruschus in the free state, bound to DNA, and in ternary complexes with associated transcription factors. These proteins orchestrate organogenesis during embryonic development, and maintain organ cells in their differentiated state in adults, suppressing carcinogenesis. Recently in our laboratories, stabilizing and destabilizing interactions have been discovered between the homeodomain and motifs in the N and C-terminal flexible random coil regions. These motif interactions appear to be modulated by phosphorylation. The stuctural mechanisms by which these interactions stabilize and destabilize the homeodomain are our current focus. Additional results tie homeodomain stability with protein turnover in the cell. Thus, these N and C-terminal motifs and their interacting kinases could be targets for therapies aimed at modulating homeodomain protein levels. In particular, increasing NKX 3-1 stability is an important therapeutic goal since lowered NKX 3-1 protein levels have been implicated in prostate tumorigenesis.[unreadable] [unreadable] 2. Electron Microscopy and Tomography Image Processing.[unreadable] [unreadable] The EMAP module in CHARMM has been developed by Dr. Wu and is becoming a very useful tool in electron microscopy structure determination. This module employs the Grid-Threading Monte Carlo method to search for the best fitting structure and is highly efficient and reduces the fitting time by one or two orders of magnitude as compared with other software available in this field. [unreadable] [unreadable] 2.1 Structure determination for Chemotaxis receptor assemblies[unreadable] [unreadable] The image processing methods developed in our lab has been successfully applied in determination of the three-dimensional structure of a chemotoaxis receptor in E-Coli cell membrane by use of electron tomography and averaging of segmented volume densities from electron tomogram. This work bridges the gap between crystallography and cryoelectron tomography of whole cells and makes a major advance in understanding of bacterial chemotaxis. Although atomic structures of both periplasmic and crytoplasmic parts of bacterial chemoreceptors have been determined, the integral structure of the entire membrane-embedded molecules remains unresolved, as does receptor arrangement in the higher-order oligomeric complexes. Our results confirm previously suggested trimer-of-dimers arrangement of receptors. In addition, we reveals two conformational states of this signal transduction machinery, providing direct structural information in the study of signal transduction mechanism.[unreadable] [unreadable] 2.2 Discovery of disordered hexagonal packing of trimeric chemoreceptor arrays in Caulobacter Crescentus. [unreadable] [unreadable] We have used cryo-electron tomography to determine the architecture of chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming signaling complexes in the cytoplasmic membrane that are packed in disordered hexagonal arrays at the cell pole. This novel packing arrangement provides an insight into the mechanisms by which cytoplasmic signaling molecules can access the receptors in activation and adaptation steps of bacterial chemotaxis.[unreadable] [unreadable] 3. Protein-protein docking with Map Objects. [unreadable] [unreadable] We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Particularly, we implemented the core-weighted correlation functions to effectively recognize correct fit of component maps in complex maps, and the grid-threading Monte Carlo search algorithm to efficiently construct complex structures from electron density maps. Dr. James M . Gruschus is applying this method in his structure study of the perioxiredoxin complex. Peroxiredoxins (Prx) are on e of several classes of proteins that reduce perioxides, reactive oxygen species produced as a by-product of cellular metabolism. For modeling the complex, the ATP-bound human Sulfiredoxin (hSrx) was docked to hyperoxidized Prx II using EMAP. In the docked structures with the Prx cysteine-sulfinic residue closest to the hSrx reactive cysteine, Asn186 of Prx II is in contact with the hSrx-bound ATP beta and gamma phosphate groups.[unreadable] [unreadable] 4. Xray Crystallography[unreadable] [unreadable] Dr. Parry has reported the structure of the class II major histocompatibility complex DRA, DRB3 in complex with an integrin peptide (PDB code 2Q6W). This is the first structure from the DRB3 gene locus. The structure models at high resolution the molecular basis of severe blood diseases associated with pregnancy, neonates, blood transfusion, stem cell and tissue transplant. A second structure is a complex with an altered ligand has been refined and awaits submission.